There is a strong driving force to develop new reliable in vitro tests to reduce the number of animal tests both in the environmental research area as well as in the drug industry. There are a number of in vitro systems developed for simulating toxic or therapeutic exposures using cultured cell systems. Some systems are developed for simulating skin, while other systems are developed to simulate the gastrointestinal canal and the respiratory tract. The respiratory tract is perhaps the most complicated to simulate, since the cultured airway epithelial cells should be exposed directly from the air with only a thin layer of liquid covering the cell surface; a so called air lifted condition. This is a particular problem if the cells shall be exposed to airborne particles. One postulation for repetitive exposure is a deagglomerated particle aerosol generated in small volumes, which is provided with the technology according to the U.S. Pat. No. 6,003,512 developed by Inhalation Sciences Sweden AB (ISAB). ISAB has also developed a spray-drying technique (WO 2004/041900) which can quickly reformulate small portions of substance to a fine powder in high yield, to be used in the dustgun machine. The dust gun aerosol generator has showed to be a useful tool to combine with an isolated, ventilated, and perfused lung of rodents (IPL) as is disclosed in P. Gerde et al. Inhalation Toxicology, 2004, 16, 45-52, wherein the pulmonary deposition of diesel soot was studied. During inhalation exposures with drug candidates, deposition of a studied substance in the target region of the lungs is a desired process that is always accompanied by unwanted losses of study material through deposition in delivery equipment and in non-target regions of the respiratory tract. Early in the development process of drugs intended for inhalation delivery, substance loss is a critical factor preventing often decisive early tests via inhalation. Two major mechanisms lead to wall deposition of particles when an aerosol is flowing through a duct: aerodynamic losses and electrostatic losses. Aerodynamic losses are contributed by diffusion, sedimentation, impaction, and interception of particles and are influenced primarily by the mass median aerodynamic diameter (MMAD) of the studied aerosol and by the fluid dynamics of the duct system under study. Aerodynamic losses can be reasonably well predicted with theoretical models. Electrostatic losses are superimposed on the aerodynamic losses, and depend strongly on material properties of the studied powder and the walls of the duct system. Electrostatic losses are more erratic and difficult to predict, and can be the dominating deposition mechanism of a studied aerosol. This unpredictable behavior of powder aerosols leads to substance losses not only to vessel walls of aerosol generator equipment, exposure systems, and to non-target regions of the respiratory tract, but also during the adjustment of exposure parameters needed to attain target exposures in study subjects. It is the intent of the current invention to address these problems. Electrostatic methods have also been devised to improve aerosol deposition in cell culture. However, these methods are more suitable for very dilute aerosols of nano-sized particles, often of air pollution origin, and are not suitable for denser pharmaceutical aerosols of clinical relevance.
There are several available methods for studying cultured airway cells are e.g. special culture wells developed to grow lung cells on top of a permeable membrane and a reservoir of perfusate under the membrane to create close contact between the airway epithelium and the surrounding air, similar to the in vivo situation. Corning has developed the “Transwell system”, where confluent and even ciliated epithelial cells may be cultured and used in experiments. There are similar systems available from Becton, Dickinson and Company; Falcon™. The company Epithelix provides ready to use, fully differentiated epithelial cells with cilia function cultured in Transwell wells, i.e., MucilAir, but they do not have a method for carrying out controlled exposures of such cells to respirable aerosols. The company Vitrocell provides cultivation- and exposure system that consists of a cell culture module based on the Transwell system and proprietary aerosol exposure units. This system accomplishes deposition of particles on a surface by the perpendicular impaction of an aerosol-laden stream onto the study surface. However, the main drawbacks with the methods available today are; lack of suitable method to create a homogenous aerosol with sufficient concentration and correct particle size distribution of the compound to be tested, and finally; poor dose control. There is certainly a need for improvement of in vitro based methods in this field. Such improvements can be accomplished by providing a well controlled method suitable for screening cell cultures for influence by all kinds of airborne particles; e.g. toxicity tests, drugs, diagnostics, metabolic studies, where also influence on the development- or differentiation or gene regulation can be studied (gene regulation studies) etc.
The invention as described in following sections obviates at least some of the disadvantages in this technology and provides a screening model that makes it possible to better study the controlled exposure of an aerosol on a model material resembling the air-blood-barrier in the respiratory tract, in vitro.